www.nature.com/scientificreports OPEN Morphological and physicochemical diversity of snow algae from Alaska Marta J. Fiołka1*, Nozomu Takeuchi2, Weronika Sofńska‑Chmiel3, Sylwia Mieszawska1 & Izabela Treska1 Snow algae are photosynthetic microbes growing in thawing snow. They usually show various morphological cell types. The aim of this study was to carry out microscopic and spectroscopic analysis of diferent forms of cells of snow algae collected on glaciers in Alaska. Four diferent shapes of algal cells were observed with the use of bright feld LM (Light Microscopy), DIC (Diferential Interference Contrast), EDF (Extended Depth Focus), fuorescence microscopy, and SEM (Scanning Electron Microscopy). The cells exhibited the strongest autofuorescence after the exposure to 365‑nm excitation light, and the intensity difered among the cell types. Zygotes (cysts) showed the most intense fuorescence. Acridine orange staining revealed the acid nature of the algal cells. The use of Congo red and Calcofuor white fuorochromes indicated diferences in the structure of polysaccharides in the cell wall in the individual types of algal cells. FTIR (Fourier‑Transform Infrared Spectroscopy) analyses showed the presence of polysaccharides not only in the algal cells but also in the fxative solution. The presence of polysaccharides in the extracellular algal fraction was confrmed by X‑ray dispersion spectroscopy (EDS), X‑ray photoelectron spectroscopy (XPS), and scanning electron microscopy imaging (SEM). The diferences observed in the structure of the cell wall of the diferent forms of red snow algae prompt further analysis of this structure. Te red or pink color of snow caused by blooms of snow algae is commonly observed during summer in alpine and coastal polar regions. It is also called watermelon snow, as it not only has the color of watermelon fesh but also emits a delicate watermelon aroma when crushed1. Snow algae have long been the subject of interest and intensive research conducted by many scientists on all continents 2. Te red color of algae is associated with the carotenoid astaxanthin found in algal cells outside chloroplasts. Tis secondary pigment probably protects photosynthetic pigment-protein complexes against damage by strong solar radiation3–6. Snow algae can cause reduction of snow surface albedo. Albedo is a ratio of the refected radiation to the total incoming solar radiation at the snow surface; thus, albedo reduction induces higher snow melting rates. Te abundance of red-pigmented algae may be associated with solar insolation, as shown by the dominance of red-pigmented algal cells in strongly insolated sites. It is assumed that the intensity of red pigmentation in algal cells is also afected by the availability of nutrients4,7. Red snow algae live in a layer of liquid water covering snow grains or between grains. Te concentration of cells in colored snow fuctuates and the intense red color indicates their high concentration8,9. Te conditions of the habitat where snow algae grow are extreme in terms of temperature. Tese microorganisms have a unique ability to live actively at around 0 °C even with very low nutrients 6,10,11. Some forms of development have thick- ened cell walls and can have an additional outer mucous coating. Tanks to these adaptations, these forms can survive dry and warm periods, i.e. afer snowmelt6. Algae involved in the phenomenon of red snow represent various species; however, most ofen, Chla- mydomonas nivalis is responsible for the red snow. Tis snow alga was described as dominant in the algal com- munity colonizing Gulkana Glacier and Harding Icefeld in Alaska13,14. Te red snow appearing in the snow area along the snow line on the glaciers can be revealed by analysis of satellite images 13. Chlamydomonas nivalis has recently been described as a member of a new genus Sanguina with two species: S. nivaloides and S. aurantia15. Cysts of diferent species of snow algae can occur together in the same area and can spread locally in the same polar regions each year2. Te difculties in identifying snow algae make the taxonomy of these microorganisms a subject of intense molecular research16. Sanguina spp. (formerly Chlamydomonas nivalis) occurs in various areas reaching the border of snow that never completely melts. Interpretations of experiments on feld-collected cells are limited due to their short-term 1Department of Immunobiology, Institute of Biology Sciences, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland. 2Department of Earth Sciences, Graduate School of Science, Chiba University, Chiba, Japan. 3Analytical Laboratory, Institute of Chemical Sciences, Maria Curie-Skłodowska University, Lublin, Poland. *email: [email protected] Scientifc Reports | (2020) 10:19167 | https://doi.org/10.1038/s41598-020-76215-x 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1. Red snow algae on Gulkana Glacier (A) and on Harding Icefeld (B) in Alaska. Te photos were taken on: (A) August 5, 2015; (B) August 9, 2015. nature, and environmental conditions occurring in nature, where there is a high variability of abiotic conditions, are hard to simulate. Nevertheless, such attempts should be made to explore these species. Sanguina spp. spend most of their life cycle as cyst cells, which are more resistant to harsh conditions. Tese forms do not divide on the snow surface throughout the summer season but show metabolic processes. Transformation into cyst cells goes along with the accumulation of storage metabolites such as pigments, lipids, and sugars 17,18. As shown by studies of snow algal blooming, algal cells are ofen found in this form19. Te analyzed microalgae have become interesting from the biotechnological point of view, as they are a source of thermostable enzymes, and have gained commercial interest as a source of astaxanthin20. Snow algae may be a potential source of pharmaceuticals, dietary supplements, or cosmetic products 21. Cultivation of microalgae creates opportunities for the production of food and fuel commodities, but the low growth rate of many species can hinder this process 22. In the era of global warming and the rapid melting of snow and ice, snow algae are of particular interest to scientists, as they contribute to environmental changes on Earth. Snow algae are an inter- esting research object for ecologists, climate scientists, microbiologists, and chemists. Tey are morphologically interesting, diverse in forms, not fully understood, and still very intriguing. Te aim of our research was to carry out morphological and physicochemical analysis of various morphologi- cal types of red snow algal cells from selected sites in Alaska using microscopic and spectroscopic techniques. Te diferent types of algal life cycle were visualized using various light and scanning electron microscopy tech- niques and compared. Te presence of intracellular and extracellular polysaccharides in the algae was examined by spectroscopic analyses as well. Materials and methods Samples. Red snow samples from two glaciers in Alaska (USA) were analyzed in this study. Te samples were collected with a stainless-steel scoop (1–2 cm in depth), melted, and preserved in a 3% formalin solu- tion (Fujiflm Wako Chemicals) in 30-ml polyethylene bottles. Te study sites were Gulkana Glacier [position: N63.28697, E145.39575, elevation, 1796 (m)] and Harding Icefeld [position: N60.17649 E149.73234, elevation, 1073 (m)]. Samples were obtained from August 5 th to 9th, 2015. Images of the red snow from Gulkana Glacier and Harding Icefeld are shown in Fig. 1. Te pH of the collected snow was 6.3–6.4. Te algal cell concentration in the snow was 3.2 ± 1.9 × 104 cells mL−1 (Gulkana Glacier) and 5.2 ± 2.6 × 104 cells mL−1 (Harding Icefeld). Light microscopy. Bright feld light microscopy, DIC microscopy (Diferential Interference Contrast), and EDF microscopy (Extended Depth of Focus) were used for imaging the algal cells. 2 µl of the cell suspension were applied to the microscope slide. Te cells were observed under a Zeiss Axiovert 40CFL light microscope Scientifc Reports | (2020) 10:19167 | https://doi.org/10.1038/s41598-020-76215-x 2 Vol:.(1234567890) www.nature.com/scientificreports/ Dye reagent Excitation wavelength (nm) Color of fuorescence Visualized structures Congo red 543 Red Cell wall polysaccharides Acridine orange 470 Green (cytoplasm), orange (acidic compartments) Cell compartments with acidic pH Calcofuor white 456 Blue Cell wall cellulose Table 1. Fluorochromes used to dye algal cells. Carl Zeiss (Germany). EDF microscopy photos were taken with an MA200 Nikon (Japan) optical microscope with inverted optics equipped with a confocal attachment that works with three lasers. 400–600 cells of each form of algal cells were measured using the ImageJ application (National Institute of Health, USA). Proflometric analysis of algal cells. Optical proflometry is a method for precise spatial imaging of the surface microgeometry of selected materials. Te surface texture, especially its roughness and waviness, is char- acterized by many parameters, the most important of which are the amplitude-height parameters. Te optical proflometer facilitates quick and contactless reproduction of surfaces in three dimensions at a low magnifca- tion. Te proflometric analysis of algal cells preserved in a 3% formalin solution was carried out using a Contour GT optical proflometer from Bruker (Germany). Te tests were performed in VSI (Vertical Scanning Interfer- ometry) mode. Te analyses were carried out with a validated method. Tree repeats of measurements were conducted for each cell type. Te roughness parameters of the four types of algal cells are presented in Table 4. SEM of algal cells. Scanning electron microscopy (SEM) was used to visualize the morphology of the algal cells more accurately. Cells preserved in a 3% formalin solution were washed in 0.1 M phosphate bufer pH 7.0, fxed with 4% glutaraldehyde, washed again in phosphate bufer, incubated in a 1% OsO4 solution for 1 h, and dehydrated with an acetone gradient (successively at 15%, 30%, 50%, 70%, and 100% for 20 min).